The present invention relates to therapeutic compositions and methods for treating and preventing infection by an immunodeficiency virus, particularly HIV infection, using chemokine proteins, nucleic acids and/or derivatives or analogs thereof.
Human immunodeficiency virus (HIV) induces a persistent and progressive infection leading, in the vast majority of cases, to the development of the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983, Science 220: 868-870; Gallo et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503) and HIV-2 (Clavel et al., 1986, Science 233:343-346; Guyader et al., 1987, Nature 326:662-669). In humans, HIV replication occurs predominantly in CD4+ T lymphocyte populations, and HIV infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-press, pp. 949-956). Retroviruses are small enveloped viruses that contain a single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439). Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-1,-II,-III), and feline leukemia virus.
The HIV viral particle consists of a viral core, composed in part of capsid proteins designated p24 and p18, together with the viral RNA genome and those enzymes required for early replicative events. Myristylated gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane. The HIV envelope surface glycoproteins are synthesized as a single 160 kilodalton precursor protein, which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammerskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV, like other enveloped viruses, introduces viral genetic material into the host cell through a viral envelope mediated fusion of viral and target membranes. HIV is targeted to CD4+ cells because a CD4 cell surface protein (CD4) acts as the cellular receptor for the HIV-1 virus (Dalgleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD4 receptor molecules (Pal et al., 1993, Virology 194:833-837; McDougal et al., 1986, Science 231:382-385; Maddon et al., 1986, Cell 47:333-348), explaining HIV""s tropism for CD4+ cells, while gp41 anchors the envelope glycoprotein complex in the viral membrane. The binding of gp120 to CD4 induces conformational changes in the viral glycoproteins, but this binding alone is insufficient to lead to infection (reviewed by Sattentau and Moore, 1993, Philos. Trans. R. Soc. London (Biol.) 342:59-66).
Studies of HIV-1 isolates have revealed a heterogeneity in their ability to infect different human cell types (reviewed by Miedema et al., 1994, Immunol. Rev. 140:35-72). The majority of extensively passaged laboratory strains of HIV-1 readily infect cultured T cell lines and primary T lymphocytes, but not primary monocytes or macrophages. These strains are termed T-tropic. T-tropic HIV-1 strains are more likely to be found in HIV-1 infected individuals during the late stages of aids (Weiss et al., 1996, Science 272:1885-1886). The majority of primary HIV-1 isolates (i.e., viruses not extensively passaged in culture) replicate efficiently in primary lymphocytes, monocytes and macrophages, but grow poorly in established T cell lines. These isolates have been termed M-tropic. The viral determinant of T- and M-tropism maps to alterations in the third variable region of gp120 (the V3 loop)(Choe et al., 1996, Cell 85:1135-1148; Cheng-Mayer et al., 1991, J. Virol. 65:6931-6941; Hwang et al., 1991, Science 253:71-74; Kim et al., 1995, J. Virol., 69:1755-1761; and O""Brien et al., 1990. Nature 348:69-73). The characterization of HIV isolates with distinct tropisms taken together with the observation that binding to the CD4 cell surface protein alone is insufficient to lead to infection, suggest a requirement for cell-type specific cofactors, in addition to CD4, for HIV-1 entry into the host cell.
Recently, certain chemokines produced by CD8+ T cells have been implicated in suppression of HIV infection. The chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage-inflammatory protein-1xcex1 and -1xcex2 (MIP-1xcex1 and MIP-1xcex2, respectively), which are secreted by CD8+ T cells, were shown to suppress HIV-1 p24 antigen production in cells infected with HIV-1 or HIV-2 isolates in vitro (Cocchi et al., 1995, Science 270:1811-1815). Additionally, high levels of these chemokines have been found to be secreted by CD4+ T lymphocytes in individuals that have been exposed to HIV-1 on multiple occasions but, remain uninfected (Paxton et al., 1996, Nature Med. 2:412-417). While RANTES, MIP-1xcex1 and MIP-1xcex2 alone or in combination, potently suppress a variety of primary HIV-1 isolates and macrophage tropic isolates, such as HIV-1BaL, some established laboratory strains, such as HIV-1IIIB, are refractory to inhibition of infection or replication by these chemokines (Cocchi et al., 1995, Science 270:1811-1815).
Chemokines, or chemoattractant cytokines, are a subgroup of immune factors that mediate chemotactic and other pro-inflammatory phenomena (See, Schall, 1991, Cytokine 3:165-183). Chemokines are small molecules of approximately 70-80 residues in length and can generally be divided into two subgroups, xcex1 which have two N-terminal cysteines separated by a single amino acid (CxC) and xcex2 which have two adjacent cysteines at the N terminus (CC). RANTES, MIP-1xcex1 and MIP-1xcex2 are members of the xcex2 subgroup (reviewed by Horuk, R., 1994, Trends Pharmacol. Sci, 15:159-165; Murphy, P. M., 1994, Annu. Rev. Immunol., 12:593-633). The amino terminus of the xcex2 chemokines RANTES, MCP-1, and MCP-3 have been implicated in the mediation of cell migration and inflammation induced by these chemokines. This involvement is suggested by the observation that the deletion of the amino terminal 8 residues of MCP-1, amino terminal 9 residues of MCP-3, and amino terminal 8 residues of RANTES and the addition of a methionine to the amino terminus of RANTES, antagonize the chemotaxis, calcium mobilization and/or enzyme release stimulated by their native counterparts (Gong et al., 1996 J. Biol. Chem. 271:10521-10527; Proudfoot et al., 1996 J. Biol. Chem. 271:2599-2603). Additionally, xcex1 chemokine-like chemotactic activity has been introduced into MCP-1 via a double mutation of Tyr 28 and Arg 30 to leucine and valine, respectively, indicating that internal regions of this protein also play a role in regulating chemotactic activity (Beall et al., 1992, J. Biol. Chem. 267:3455-3459).
The monomeric forms of all chemokines characterized thus far share significant structural homology, although the quaternary structures of xcex1 and xcex2 groups are distinct. While the monomeric structures of the xcex2 and xcex1 chemokines are very similar, the dimeric structures of the two groups are completely different. An additional chemokine, lymphotactin, which has only one N terminal cysteine has also been identified and may represent an additional subgroup (xcex3) of chemokines (Yoshida et al., 1995, FEBS Lett. 360:155-159; and Kelner et al., 1994, Science 266:1395-1399).
Receptors for chemokines belong to the large family of G-protein coupled, 7 transmembrane domain receptors (GCR""s) (See, reviews by Horuk, R., 1994, Trends Pharmacol. Sci. 15:159-165; and Murphy, P. M., 1994, Annu. Rev. Immunol. 12:593-633). Competition binding and cross-desensitization studies have shown that chemokine receptors exhibit considerable promiscuity in ligand binding. Examples demonstrating the promiscuity among xcex2 chemokine receptors include: CC CKR-1, which binds RANTES and MIP-1xcex1(Neote et al., 1993, Cell 72: 415-425), CC CKR-4, which binds RANTES, MIP-1xcex1, and MCP-1 (Power et al., 1995, J. Biol. Chem. 270:19495-19500), and CC CKR-5, which binds RANTES, MIP-1xcex1, and MIP-1xcex2 (Alkhatib et al., 1996, Science, in press and Dragic et al., 1996, Nature 381:667-674). Erythrocytes possess a receptor (known as the Duffy antigen) which binds both xcex1 and xcex2 chemokines (Horuk et al., 1994, J. Biol. Chem. 269:17730-17733; Neote et al., 1994, Blood 84:44-52; and Neote et al., 1993, J. Biol. Chem. 268:12247-12249). Thus the sequence and structural homologies evident among chemokines and their receptors allows some overlap in receptor-ligand interactions.
CC CKR-5 is the major coreceptor for macrophage-tropic strains of HIV-1 (Alkhatib et al., 1996, Science, in press; Choe et al., 1996, Cell 85:1135-1148; Deng et al., 1996, Nature 381:661-666; Doranz et al., 1996, Cell 85:1149-1158; Dragic et al., 1996, Nature 381:667-674). RANTES, MIP-1xcex1, or MIP-1xcex2, the chemokine ligands for this receptor block HIV Env-mediated cell fusion directed by CC CKR-5 (Alkhatib et al., 1996, Science, in press; and Dragic et al., 1996, Nature 381:667-674). Additional support for the role of CC CKR-5 as an M-tropic HIV-1 cofactor comes from the finding that a 32-base pair deletion in the CC CKR-5 gene found in three multiply exposed but uninfected individuals, prevents HIV from infecting macrophages (Liu et al., 1996, Cell 86:367-377). However, only three of the 25 uninfected individuals studied had this mutation.
The V3 loop of gp120 is the major determinant of sensitivity to chemokine inhibition of infection or replication (Cocchi et al., 1996, Nature Medicine 2:1244-1247; and Oravecz et al., 1996, J. lmmunol. 157:1329-1332). Signal transduction through xcex2 chemokine receptors is not required for inhibition of HIV infection or replication, since RANTES inhibits HIV-1 infection in the presence of pertussis toxin, an inhibitor of G-protein-mediated signaling pathways (P. M. Murphy 1994, Ann. Rev. Immunol. 12:593-633; Bischoff et al., 1993, Eur. J. Immunol. 23:761-767; and Simon et al., 1991, Science 252:802-807). CxC CKR4, a CxC (xcex1) chemokine receptor, has been shown to be a coreceptor involved in infection by laboratory-adapted HIV-1 strains (Fong et al., 1996, Science 272:872-877). The xcex1 chemokine SDF-1, the ligand for this receptor, has been demonstrated to block infection by T-tropic HIV-1 isolates. CxC CKR4 does not bind the beta chemokines RANTES, MIP-1xcex1, or MIP-1xcex2.
Recently, it has been shown that certain primary, syncytium-inducing/T-tropic isolates use both CC CKR5 and CxC CKR4 as coreceptors and are able to switch between the two. Thus, in the presence of RANTES, MIP-1xcex1 and MIP-1xcex2, the chemokine ligands for CC CKR5, T-tropic strains are still able to infect cells via the CxC CKR4 coreceptor (Zhang et al., 1996, Nature 383:768).
HIV infection is pandemic and HIV-associated diseases represent a major world health problem. Although considerable effort is being put into the design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya et al., 1991, FASEB J. 5:2369-2381). Many viral targets for intervention with the HIV life cycle have been suggested, as the prevailing view is that interference with a host cell protein would have deleterious side effects. For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2N,3N-dideoxynucleoside analogs such as AZT, ddI, ddc, and d4 T have been developed which have been shown to been active against HIV (Mitsuya et al., 1991, Science 249:1533-1544).
The new treatment regimens for HIV-1 show that a combination of anti-HIV compounds, which target reverse transcriptase (RT), such as azidothymidine (AZT), lamivudine (3TC), dideoxyinosine (ddi), dideoxycytidine (ddc) used in combination with an HIV-1 protease inhibitor have a far greater effect (2 to 3 logs reduction) on viral load compared to AZT alone (about 1 log reduction). For example, impressive results have recently been obtained with a combination of AZT, ddI, 3TC and ritonavir (Perelson et al., 1996, Science 15:1582-1586). However, it is likely that long-term use of combinations of these chemicals will lead to toxicity, especially to the bone marrow. Long-term cytotoxic therapy may also lead to suppression of CD8xe2x88x92 T cells, which are essential to the control of HIV, via killer cell activity (Blazevic et al., 1995, AIDS Res. Hum. Retroviruses 11:1335-1342) and by the release of factors which inhibit HIV infection or replication, notably the chemokines Rantes, MIP-1xcex1 and MIP-1xcex2 (Cocchi et al., 1995, Science 270:1811-1815). Another major concern in long-term chemical anti-retroviral therapy is the development of HIV mutations with partial or complete resistance (Lange, J. M., 1995, AIDS Res. Hum. Retroviruses 10:S77-82). It is thought that such mutations may be an inevitable consequence of anti-viral therapy. The pattern of disappearance of wild-type virus and appearance of mutant virus due to treatment, combined with coincidental decline in CD4+ T cell numbers strongly suggests that, at least with some compounds, the appearance of viral mutants is a major underlying factor in the failure of AIDS therapy.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection, by focusing on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley et al., 1990, Ann. Int. Med. 112:247-253; Kahn et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific processing of certain viral encoded proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533). The clinical outcome of these candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin et al., 1985. Science 228:1094-1096). Thus far, therefore, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. Several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff et al., U.S. Pat. No. 5,141,867; Saith et al., WO 92/22654; Shafferman, A., WO 91/09872; Formoso et al., WO 90/07119. To this end, vaccines directed against HIV proteins are problematic in that the virus mutates rapidly rendering many of these vaccines ineffective. Clinical results concerning these candidate vaccines, however, still remain far in the future.
Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, effective, non-toxic treatments are still needed.
Citation of a reference hereinabove shall not be construed as an admission that such reference is prior art to the present invention.
The present invention relates to prophylactic and therapeutic methods and compositions based on chemokine proteins, nucleic acids, derivatives or analogs thereof that inhibit replication and/or infection of an immunodeficiency virus in vitro or in vivo, decrease viral load, and/or treating or preventing diseases and disorders associated with infection of an immunodeficiency virus. In specific embodiments, the immunodeficiency virus inhibited by the methods and compositions of the invention is HIV.
According to the present invention, different chemokine receptors are involved in immunodeficiency virus infection, depending on the particular isolate. The present invention provides methods of identifying the particular chemokine(s) capable of inhabiting the infection or replication of a viral isolate of a particular patient and of treating such patient. Pharmaceutical compositions comprising chemokines heretofore unknown to be active against HIV are also provided, as well as related methods of treatment or prophylaxis.
The invention also relates to chemokine derivatives or analog(s) that bind to a plurality of chemokine receptors and that are effective at preventing diseases or disorders associated with infection of an immunodeficiency virus, particularly HIV infection. The invention also relates to pharmaceutical compositions containing such therapeutically and prophylactically effective chemokine derivatives or analogs, or the nucleic acids encoding such. In one embodiment, the chemokine derivative or analog binds to one or more xcex2 chemokine receptors selected from a group consisting of CC CKR-1, CC CKR-2A, CC CKR-2B, CC CKR-3, CC CKR-4 and CC CKR-5. In a preferred embodiment, the derivative or analog binds to the chemokine receptor CC CKR-5. In another embodiment, the chemokine derivative or analog binds to one or more a chemokine receptors selected from the group consisting of CxC CKR4, IL-8RA, IL-8RB, Mig receptor, xcex3IP-10 receptor, and Duffy antigen. In a preferred embodiment, the derivative or analog binds to both an xcex1 chemokine receptor and a xcex2 chemokine receptor. In a most preferred embodiment, the derivative or analog binds to both CxC CKR4 and CC CKR-5. In another embodiment, the chemokine derivative or analog binds to 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 chemokine receptors.
The present invention also relates to pharmaceutical compositions comprising one or more xcex1, xcex2, or xcex3 chemokines, or nucleic acids encoding the foregoing, in an amount effective to inhibit HIV infection or replication. In one embodiment, the pharmaceutical compositions of the invention comprise RANTES, MIP-1xcex1, MIP-1xcex2, MCP-1, MCP-3 or IL-8 nucleic acid encoding RANTES, MIP-1xcex1, MIP-1xcex2, MCP-1, MCP-3 or IL-8 or a therapeutically and prophylactically effective derivative or analog thereof or nucleic acid encoding the same, in combination with another chemokine, nucleic acid encoding another chemokine, or derivative or analog thereof, in an amount effective to treat or prevent diseases or disorders associated with immunodeficiency virus infection, particularly HIV infection, e.g., ARC, AIDS. In another embodiment, the pharmaceutical composition comprises a xcex2 chemokine, or nucleic acid encoding a xcex2 chemokine, selected from the group consisting of MCP-2, MCP-4, MIP-1xcex3, MIP-3xcex1, MIP-3xcex2, eotaxin, Exodus, and I-309, MIP-3xcex1, MIP-3xcex2, eotaxin, Exodus, or a therapeutically or prophylactically effective derivative or analog thereof. In an additional embodiment, the pharmaceutical composition comprises an a chemokine, nucleic acid encoding an xcex1 chemokine, or therapeutically or prophylactically effective derivative or analog thereof. In a further embodiment, the pharmaceutical composition comprises the xcex3 chemokine lymphotactin, nucleic acid encoding lymphotactin, or a therapeutically or prophylactically effective derivative or analog thereof. In one embodiment, the pharmaceutical composition of the invention comprises an xcex1 chemokine, or nucleic acid encoding an xcex1 chemokine, selected from the group consisting of xcex3IP-10, PF4, NAP-2, GRO-xcex1, GRO-xcex2, GRO-xcex3, ENA-78, GCP-2, or a therapeutically effective derivative or analog thereof. In yet another embodiment, the pharmaceutical composition of the invention contains a combination of xcex1, xcex2 and/or xcex3 chemokines, nucleic acids encoding xcex1,xcex2 and/or xcex3 chemokines, or therapeutically or prophylactically effective derivatives or analogs thereof.
The present invention also relates to therapeutic compositions based on chemokines and nucleic acids encoding chemokines. Therapeutic compounds of the invention include but are not limited to chemokines, nucleic acids encoding chemokines, and derivatives (including fragments and chimerics) and analogs thereof, that are effective at inhibiting replication or infection by an immunodeficiency virus.
The invention further relates to therapeutic methods for treatment and prevention of diseases and disorders associated with infection with an immunodeficiency virus, in particular HIV infection, by administering a therapeutic composition of the invention. More specifically, the invention provides methods for formulating and administering pharmaceutical compositions of the invention that inhibit infection or replication of one or more known isolates of an immunodeficiency virus, preferably of HIV.
The invention further provides methods for inhibiting the infection or replication of an immunodeficiency virus isolate, in particular, an HIV isolate. In a preferred embodiment, the invention provides methods for formulating, on a patient-to-patient basis, a therapeutic composition of the invention for treating diseases and disorders associated with the immunodeficiency virus isolate(s) present in an individual at a given time. Methods for administering the prophylactic or therapeutic compositions of the invention are also provided.
The invention further provides methods for treating or preventing diseases and disorders associated with infections by immunodeficiency viruses, particularly HIV infections, comprising administering a pharmaceutical composition of the invention containing one or more therapeutically and/or prophylactically effective chemokine derivative(s) and/or analog(s) that bind to a plurality of chemokine receptors. Methods for identifying such derivatives or analogs and formulating the prophylactic or therapeutic compositions are also provided.
In a preferred embodiment, the invention relates to a pharmaceutical composition comprising MDC and I-309. In a related aspect, the invention relates to a method for treating HIV infection, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of MDC (and/or analogs. and/or derivatives thereof) and I-309 (and/or analogs and/or derivatives thereof). The MDC (and/or analogs and/or derivatives thereof and I-309 (and/or analogs and/or derivatives thereof) may be administered simultaneously or sequentially. Moreover, the MDC (and/or analogs and/or derivatives thereof) and I-309 (and/or analogs and/or derivatives thereof) are suitably administered together as components of a pharmaceutical composition, along with a pharmaceutically acceptable carrier. The components are preferably administered in a synergistic amount and in a therapeutically effective amount.
A xe2x80x9ctherapeutically effectivexe2x80x9d amount or dose is an amount or dose which prevents or delays the onset or progression of an indicated disease or other adverse medical condition. The term also includes an amount sufficient to arrest or reduce the severity of an ongoing disease or other adverse medical condition, and also includes an amount necessary to enhance normal physiological functioning.
As used herein, xe2x80x9ctreatmentxe2x80x9d of a disease or other adverse medical condition, should be broadly interpreted based on the therapeutic effects described herein as variously including palliative, active, causal, conservative, medical, palliative, prophylactic, and/or symptomatic treatment, treatment designed to delay the onset or progression of the disease or other adverse medical condition, as well as treatment designed to arrest or reducing the severity of an ongoing disease or other adverse medical condition.
As used herein, a xe2x80x9cpharmaceutically acceptablexe2x80x9d component (such as a salt, carrier, excipient or diluent) of a formulation according to the present invention is a component which (1) is compatible with the other ingredients of the formulation in that it can be combined with the therapeutics of the invention without eliminating the biological activity of the therapeutics; and (2) is suitable for use in non-human animals or humans without undue adverse side effects (e.g., toxicity, irritation, and allergic response). Side effects are xe2x80x9cunduexe2x80x9d when their risk outweighs the benefit provided by the pharmaceutical composition.
As used herein, a xe2x80x9cpharmaceutically acceptablexe2x80x9d with reference to the degree of purity of a polypeptide (e.g., a chemokine or chemokine analog or chemokine fragment) or nucleic acid indicates that the polypeptide or nucleic acid (1) is free of contaminating materials that would eliminate the biological activity of the polypeptide or nucleic acid; and (2) is free of contaminating materials that would render the therapeutic (e.g., polypeptide or nucleic acid) unsuitable for administration to humans (for pharmaceutical use) or other animals (for veterinary use) by causing undue adverse side effects (e.g., toxicity, irritation, and allergic response). Side effects are xe2x80x9cunduexe2x80x9d when their risk outweighs the benefit provided by the therapeutic (e.g., polypeptide or nucleic acid).
The term xe2x80x9csubstantially purexe2x80x9d when used in reference to a polypeptide or nucleic acid is defined herein to mean a therapeutic (e.g., polypeptide or nucleic acid) that is substantially free from other contaminating proteins, nucleic acids, and other biologicals derived from an original source organism, recombinant DNA expression system, or from a synthetic procedure employed in the synthesis or purification of the therapeutic (e.g., chromatography reagents and polymers, such as acrylamide or agarose). Purity may be assayed by standard methods. Purity evaluation may be made on a mass or molar basis.